J Vet Sci.  2011 Jun;12(2):191-193. 10.4142/jvs.2011.12.2.191.

A simplified PCR assay for fast and easy mycoplasma mastitis screening in dairy cattle

• Higuchi H
• Iwano H
• Kawai K
• Ohta T
• Obayashi T
• Hirose K
• Ito N
• Yokota H
• Tamura Y
• Nagahata H

• KMID: 1106563
• DOI: http://doi.org/10.4142/jvs.2011.12.2.191

Abstract

A simplified polymerase chain reaction (PCR) assay was developed for fast and easy screening of mycoplasma mastitis in dairy cattle. Species of major mycoplasma strains [Mycoplasma (M.) bovis, M. arginini, M. bovigenitalium, M. californicum, M. bovirhinis, M. alkalescens and M. canadense] in cultured milk samples were detected by this simplified PCR-based method as well as a standard PCR technique. The minimum concentration limit for detecting mycoplasma by the simplified PCR was estimated to be about 2.5 x 10(3) cfu/mL and was similar to that of the standard PCR. We compared the specificity and sensitivity of the simplified PCR to those of a culture method. Out of 1,685 milk samples cultured in mycoplasma broth, the simplified PCR detected Mycoplasma DNA in 152 that were also positive according to the culture assay. The sensitivity and specificity of the simplified PCR were 98.7% and 99.7%, respectively, for detecting mycoplasma in those cultures. The results obtained by the simplified PCR were consistent with ones from standard PCR. This newly developed simplified PCR, which does not require DNA purification, can analyze about 300 cultured samples within 3 h. The results from our study suggest that the simplified PCR can be used for mycoplasma mastitis screening in large-scale dairy farms.

Keyword

bovine; mastitis; mycoplasma; simplified PCR

MeSH Terms

Animals
Cattle
Colony Count, Microbial/veterinary
DNA, Bacterial/chemistry/genetics
Disease Outbreaks/prevention & control/veterinary
Female
Mastitis, Bovine/diagnosis/*microbiology
Milk/cytology/*microbiology
Mycoplasma/genetics/*isolation & purification
Mycoplasma Infections/diagnosis/microbiology/*veterinary
Polymerase Chain Reaction/veterinary

Figure

• 

  Fig. 1 Detection of major mycoplasma mastitis pathogens by simplified (upper) and standard (lower) PCR. M. marker, 1: Mycoplasma (M.) bovis (ATCC 25523), 2: M. arginini (ATCC 23838), 3: M. bovigenitalium (ATCC 19852), 4: M. californicum (ATCC 33461), 5: M. bovirhinis (ATCC 27748), 6: M. alkalescens (ATCC 29103), 7: M. canadense (ATCC 29418).

• 

  Fig. 2 Minimum limits of detection for major mycoplasma mastitis pathogens by simplified PCR and standard PCR. 1: M. bovis (ATCC 25523), 2: M. arginini (ATCC 23838), 3: M. bovigenitalium (ATCC 19852), 4: M. californicum (ATCC 33461), 5: M. bovirhinis (ATCC 27748), 6: M. alkalescens (ATCC 29103), 7: M. canadense (ATCC 29418).

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